Hybridization assay and means to be used in the assay

ABSTRACT

A method for the detection of a nucleotide sequence of a nucleic acid in a sample. The method comprises the steps: (i) contacting under hybridization condition the single stranded form of the nucleotide sequence with a single stranded nucleic acid probe, in which plurality of rare earth metal chelate groups is covalently linked via a water-soluble polymer of non-nucleic acid structure to a nucleotide acid that as one of its strand has the nucleotide sequence to be detected and as the other strand the nucleotide sequence of the probe, and (ii) detecting the formation of double stranded nucleic acid. The plurality of rare earth metal chelate groups have at least one metal ion selected from the group consisting of Eu 3+ , Sm 3+ , Tb 3+  and Dy 3+  as the chelated rare earth metal. The probes as given above are also claimed.

BACKGROUND OF THE INVENTION

This invention relates to a hybridization assay utilizing nucleic acidprobes labelled with lanthanide chelates that show time-delayedfluorescence.

Labelled nucleic acids have become indispensable in hybridizationassays, performed both in vitro and, as in hybridocytochemicalmicroscopy, also in vivo. Appropriate labelling of nucleic acids is acrucial point in their sequencing and applications may also be found inthe different methodologies of nucleic acid separation. Nowadays, mostof the efforts to find more sensitive markers have been made in thefield of hybridization probes for the detection of specific,complementary nucleic acid sequences. This is natural in view of thegreat importance of such assays in medicine and molecular biology.

DNA or RNA can be labelled in a variety of ways. Generally, all labelsmay be detected directly i.e. the label, which is bonded to the nucleicacid is itself detectable, or indirectly when the label participates inone or more reactions thus generating detectable products.

DESCRIPTION OF PRIOR ART

Several new labelling methodologies have recently become available.Despite of many differences they can be systematized on the basis ofsome main criteria. In nucleic acid technology a common name fordifferent labels is reporter group.

1. Methods of DNA Detection

a) Direct detection method: Nucleic acids are commonly labelled with theradioisotopes ³² P, ¹²⁵ I, ³ H or fluorescent markers. Especially inroutine analyses the radioactive material tends to be replaced by thenon-radioactive labels because of the serious drawbacks associated withthe use of radioactive labels. Safety and disposal problems are obvious,but the low stability of materials with high specific activity togetherwith their high cost should not be forgotten either.

Theoretically, fluorescent compounds are ideally suited to replaceradioactive isotopes. To date the only examples of such fluorescentmarkers used in DNA labelling are fluorescein, rhodamine, Texas Red andNBD. The calculated high sensitivity which could be achieved using thistype of reagents is, however, to a great extent limited by the fact thatmost biological samples including proteins also fluoresce thus bringingthe background to a not always acceptable level to distinguish betweenthe fluorescent marker and the protein.

b) Indirect detection method: Nucleic acids are labelled with differentproteins possessing enzymatic activities e.g. alkaline phosphatase orhorse radish peroxidase. The subsequent reaction of the appropriatesubstrate catalyzed by the attached enzyme usually generates aneasy-to-detect colored product. It is constantly emphasized that anadvantage of such a system is the fact that there is no need for adetecting apparatus, but this fact can also be seen as a disadvantagesince this visual technique is not well-suited to quantitative analysis.

2.Attachment of a Detectable Group to DNA or RNA

The way in which report groups are linked to nucleic acids may serve asanother criteria of differentiating between labelling techniques.

Direct attachment means that a detectable marker is bound to a nucleicacid already before a particular analytical process takes place. Thelabel may be coupled to the nucleic acid enzymatically as inradiolabelling, by using DNA kinase and τ ³² P ATP, or by employingdifferent σ ³² P nucleoside triphosphates in the nick translationprocess. A properly activated label which is able to react with anyexisting or created function in nucleic acids, can also be chemicallyattached to nucleic acids.

Indirect attachment of the detectable group to nucleic acids can berealized by several methods. Among the most commonly used methods is thelabelling of nucleic acids with a hapten, thus rendering them detectableby immunological means.

Examples of haptens useful in indirect attachment are fluorescein and atrinitrophenyl group (TNP). Antibodies with high affinity to thesehaptens have been well-studied and are easily obtainable. However, thecomplexity of nucleic acid derivatization together with a rather complexsystem of detection, problems in purification of some of the compounds,and quite a low sensitivity of the assay, make this technique ratherunattractive.

N-acetoxy-N-2-acetaminofluorence (AAF) has been used for the chemicalmodification of nucleic acids for sensitive, colorimetric detection oftarget DNA using specific antibodies and peroxidase or alkalinephosphatase second antibody conjugates.

However, in view of the strong carcinogenic properties of AAF, othertypes of labels have been tried. For example biotin has been attachedchemically to deoxyribonucleo-tides and these nucleotides wereintroduced into DNA by nick translation. A single stranded portion ofbiotin-DNA thus has been used as a hybridization probe. To detect doublestranded DNA having biotin or hapten bound to one of its strands avidinor anti-hapten antibodies have been used. The DNA or protein is eitherlabelled (e.g. by fluorescence markers) or detected by typicalimmunological methods.

The laborious procedure for the derivatization of DNA with biotin hasbeen simplified by introducing a photo-activatable analog of biotin forthe labelling of nucleic acids. However, the number of biotin groupswhich can be introduced to the DNA molecule, is limited in both thebiotin and biotin analog methods. It has been found that the biotintechnique is extremely difficult to carry out successfully and theresults obtained are quite varied. The method is therefore extremelyunreliable and does not provide any basis for routine polynucleotidesequence detection. Therefore, the approach of cross-linking biotinlabelled histone to single stranded nucleic acids with glutaraldehyde,was useful. However, despite the possibility of a substantial increasein the biotin content, the sensitivity of assay was not sufficientlyhigh.

Some General Remarks on Known Labelling Techniques

It is obvious that in the design of hybridization probes all labellingmethods based on random derivatization of the exocyclic amino functionin nucleic acids with an appropriate label must employ polydeoxy nucleicacid. A short DNA sequence, for example an oligo nucleotide, is toosensitive for such an operation if effective further hybridization isexpected. When using a long DNA probe the hybridization temperature hasto be relatively high unless special additives (e.g. a highconcentration of DMF) are present in the test mixture. Such hightemperatures may limit the use of some enzymes as detectable markers bydecreasing their activity. Moreover, it is well known that the longerDNA sequence requires a much longer time for hybridization to proceed, afact which seriously limits the speed of assay without giving aproportionally equal increase in sensitivity.

In the literature there are only few reports of employingoligodeoxynucleotides labelled with non-radioactive markers for use ashybridization probes. The use of an octadecamer selectivelymonobiotinylated at the 5' position has proven unsuccessful as thesensitivity of the assay was far too low.

It is therefore commonly accepted that of all methods making use ofindirect labelling or indirect detectable markers, the preferredprocedure involves amplification of the signal. This is laborious,increases errors of method and makes routine analyses very difficult.

Polymeric substances of the non-nucleotide type have been employed inthe design of non-radioactive hybridization probes. Biotin-labelledhistone has been cross-linked to single-stranded nucleic acids withglutaraldehyde. The detection of target DNA with these probes andavidine-peroxidase conjugates was less sensitive than with radioactivemethods. In another strategy, peroxidase or alkaline phosphatase wascross-linked to a polyethylene-imine of low molecular weight withp-benzoquinone and the resulting conjugates were cross-linked to DNAwith glutaraldehyde. In both cases the polymers were used primarily aslinkers between the DNA probe and the detectable units. It is thereforeimportant to emphasize that in the present hybridization assay the mainobject of using a polymeric matrix is to amplify the detectable signalthus making the assay more sensitive. The polymeric matrix is not usedas a simple linker molecule.

Hybridization assays employing rare earth chelates have been describedbefore our priority date (EP-A-97,373; Syvanen et al., Nucl Acids Res14(1986) 1017-27; and Hurskainen et al., In: NOMBA Nordforsk SymposiumGene Technology in Basic and Applied Research (Abstr.) Savonalinna,Finland, May 27-29, 1984, 12). Further details of other hybridizationassays have been given during the priority year (WO-A-87/02708; andEP-A-212,951).

A survey of the literature in this area clearly shows that when the aimis to design an easy, reliable and sensitive hybridization method, it isdesirable to use directly labelled and detectable probes. This is incontrast to sandwich techniques, when amplification of signal is desiredit is generally realized by increased label density. A hybridizationassay method of choice should not involve either radioactive isotopes orhighly toxic intermediates.

The Invention

The present invention provides an improved hybridization assay methodutilizing certain labelled nucleic acid probes. It is a method for thedetection of a nucleotide sequence of a nucleic acid in a sample. Themethod has the following major characteristic steps:

(i) Contacting under hybridization condition the single stranded form ofthe nucleotide sequence present in a sample with a single strandednucleic acid probe, so as to form a double stranded nucleic acid whichhas as one of its strands the nucleotide sequence to be detected and asthe other of its strands the nucleotide sequence of the probe. The probehas as one part, a nucleotide sequence that is complementary to thesequence to be determined, and as the other, a plurality of rare earthmetal chelate groups that ar covalently linked, via a water-solublepolymer of non-nucleic acid structure, to its nucleotide sequence. Oneprobe can contain many sequences complementary to the one to bedetermined and/or of pluralities of rare earth metal chelate groups.

(ii) Determining or detecting the double stranded nucleic acid so formedby using time-resolved spectrofluorometry to measure the rare earthmetal chelate incorporated into the double stranded nucleic acid.

The plurality of rare earth metal chelate groups has at least one metalion selected from the group consisting of Eu³⁺, Sm³⁺, Tb³⁺ and Dy³⁺, andis preferably europium and terbium ions. The intensity of fluorescenceemitted from the double stranded nucleic acid is a quantitative measureof the nucleotide sequence to be determined. The use of a covalentlybound polymeric group carrying several rare earth metal chelate groupsamplifies the signal from the probe. A brief calculation of thefluorescence intensity using a probe labelled with only a fewfluorescent markers showed that the sensitivity of such an assay will beinsufficient. This realization is especially applicable to the detectionof viral DNA in the early stages of infection. Therefore, the importantfeature in the invention is the use of water soluble polymeric compoundsas a matrix to which a large number of europium or terbium chelates arecovalently coupled. This covalent coupling gives a large amplificationof the detectable signal. Contrary to many existing methodologies, thepresent hybridization assay is a straightforward, direct, and one-stepprocedure.

One important aspect of the invention is the particular probes that aredescribed in this specification and that are used in the assay of theinvention. For practical considerations as shown in the examples, thechelated lanthanide ion always non-radioactive.

Hybridization Assays

Hybridization assays are well-known in the art. As in common immunoassaytechniques, the hybridization assays can be divided into two groupsincluding homogeneous assays and heterogenous assays. The homogenoushybridization assays utilize labels that in one way or the other changetheir signal as a consequence of being incorporated into double strandedforms of DNA. Accordingly no separation of single stranded nucleic acidsfrom double stranded nucleic acids is required in the homogenous assayvariants (see for instance EP-A-144,914 and EP-A-70,685). In theheterogenous assays, separation of double stranded DNA containing theprobe from unhybridized probe is accomplished by the use of a matrixwhich is insoluble in the assay medium and which selectively is able tobind either the double stranded DNA or the unhybridized probe. Thebinding may be carried out as a biospecific absorption employingcovalently bound oligonucleotide sequences or other biospecific affinityreactants. Isolubilized streptavidin or antibodies can specificallyabsorb nucleic acids equipped with biotinyl or homologous haptenicgroups. (See for instance Dunn et al, Cell 12 (1977) 23-36; Ranki M etal., Gene 21 (1983) 77-85; Meinkoth and Wahl, Anal Biochem 138 (1984)267-84; Syvanen et al., Nucl Acids-Res 14 (1986) 5037-48; Dattagupta andCrothers, EP-A-130,523; WO-A-85/02628 and U.S. Pat. No. 4,563,419). Themost popular heterogenous method at present time is hybridization onfilter paper, e.g. nitro cellulose paper. In this hybridization assaymethod the single stranded DNA to be assayed is adsorbed to the filterpaper, and thereafter the filter paper is saturated with DNAnon-homologous to the DNA to be assayed and contacted underhybridization conditions with the DNA probe.

The conditions required to accomplish hybridization depend on the probelength, i.e. the length of the oligonucleotide sequence to behybridized, and the specificity desired. As a general rule, longernucleotide sequences that are complementary require a longer time forhybridization to occur. An increase in temperature and/or specialadditives, such as DMF, may produce more rapid reactions. Normally thehybridization solutions are buffered to pH values of 5-9 and thehybridization are carried out at constant temperatures, 18°-65° C. for3-48 hours. For homogenous variant hybridization assays it is verycritical not to add to the hybridization media, chemicals whichnegatively influence the signal emitted by the label(s).

The Polymer and Its Derivatization

The water-soluble polymer used in the present hybridization assay is ofnon-nucleic acid structure, and may be a biopolymer or a syntheticpolymer. Derivatized forms of biopolymers may also be used. When notbound to a nucleic acid, the polymer should exhibit a plurality offunctional groups allowing covalent attachment to a nucleic acid. Thusthe most suitable polymers have more than 10, such as more than 50OH-groups or amino groups. The OH-groups may be part of a carboxylicacid group or an alcoholic or phenolic hydroxyl group. The polymer canhave molecular weights above 1,000 daltons, and preferably molecularweights above 5,000 daltons. In most cases the polymer has a molecularweight below 10⁶ daltons.

Linear or substantially linear polymers are preferred because they willhave a favorable geometry in relation to the hybrids formed. Anotherobvious demand placed on such polymers is their solubility in water.Free water solubility of the polymer chelate derivatives, as well aswater solubility of the final lanthanide chelate-polymer-DNA complex areimperative.

The first successful attempt to synthesize the derived polymers of theinvention was made by condensation of polyacids with chelates possessinga free amino group. A water soluble carbodiimide was used as thecoupling reagent. Next, since most of the chelates available were bestsuited for derivatization of free, preferably primary, amino groups withrelatively high pKa (optimum region pKa 8-11), polymers which meet suchfunctional requirements were directly tested. Other polymers of interestwere prepared by appropriate derivatization, introducing free aminofunctions to the polymers which lacked them from the beginning.

Particularly good coupling results were obtained with the followingpolymers: polysaccharides, such as chemically modified dextrane,polyvinylamine (PV.A), polyethyleneimine (PEA), polylysine (PL),chemically modified polyacrylamide, carboxymetylated polyvinylamine (CMPVA) and polyacrylic acid. Two of these, polyethyleneimine and dextrane,do not fully meet the criterion of linearity. Nevertheless, bothcompounds consist of long intervals within which the required lineargeometry is preserved and accordingly are substantially linear. Allthese polymers are readily dissolved in water at all derivative stages.

Polyvinylamine was prepared according to a known method, and stored in aconvenient form of hydrochloride as dry powder. This material (M^(PS)3.4×10⁴) has been used as a matrix for the synthesis of polymeric watersoluble dyes and has been shown to be the best alternative because ofits high reactivity. It has the highest density of the groups which canbe derivatized. Polyethylene-imine used in our experiments had anaverage molecular weight of 5×10⁴ to 6×10⁴, 1,5×10⁴ to 3×10⁴. Polylysinehydrobromides used had molecular weights of 3×10⁴ to 7×10⁴,respectively. Functionalized polyacrylamide was made frompolyethylacrylate Mw 72,000. The synthesis is described in Example 1.The polyacrylic acid used had a Mw≈5,000. The procedure forcarboxymethylation of polyvinylamine is outlined as Example 2.

Polyacrylamide is an example of a desired compound which is prepared notby derivatization of already existing polymeric material but through thesynthesis of a monomeric acrylamide chelate and its subsequentpolymerization as in Example 3. Copolymerization of such monomers withother appropriate acrylamide derivatives will create products possessingall the necessary features like proper solubility, net charge, and thepresence of other functional groups.

Theoretically, two different derivatization methods are possible forpolymers possessing free amino functions. Derivatization with alanthanide chelate followed by a derivatization with an appropriatebifunctional reagent, and coupling of an activated oligo-DNA or poly-DNAprobe is the most economical derivatization method. Reverse order of thereactions is also possible using this method since the reactivity of theexocyclic amino function in DNA is very low, and thus no derivatizationwith chelate takes place on these amino functions. This has beenverified in several blank reactions with DNA and the active forms ofchelates.

For acidic polymers (polymers containing for instance carboxylic acidgroups) only the first route, i.e. functionalization with a chelate,followed by reaction with a bifunctional coupling reagent and theaddition of nucleic acid can be used.

Both in case of polyamines and polyacids polymers, the reaction cyclecould be stopped at the level of an activated polychelate (Examples 4and 5). Such functionalized polymers can be stored for long period oftime and coupled with different DNA probes whenever necessary. This typeof derivatization has therefore the advantage of being "universal".

Functionalization of acidic polymers is a very quick process, and thedegree of condensation, performed in a water solution and employing alarge excess of EDAC (water-soluble condensing agent), was directlyproportional to the amount of used amino reagent.

The labelling of amino-functionalized polymers with chelates has beenperformed in aqueous media using a triethylammonium bicarbonate buffer,pH 10.

The use of a phosphate buffer should be avoided as it forms insolublesalts in water with polyamines. The extent of the derivatization caneasily be manipulated by changing the pH or the chelate concentration.For instance, using two equivalents of isothiocyanato chelate per eachamino group in PVA at pH 10, essentially all amino functions can bederivatized in an overnight reaction.

By performing this reaction at pH 7, only 35-50% of the amino groupswere labelled. This was determined after standard gel filtration andcounting of lanthanide fluorescence.

The Nucleic Acid Portion of the Probe

Two general alternatives exist in the choice of the nucleic acid portionof the probe.

The use of polynucleotides is often favorable when the nucleic acid iseasily accessible and no data exists on the sequence of the targetmolecule. The probe can be prepared e.g. from a double-stranded DNAidentical in sequence to two strands of the gene sequence to bedetected. On denaturation of the double-stranded DNA, the two strandsare hybridized to two strands of the gene sequence to be detected. Theprobing sequence of its double-stranded precursor can be produced bycloning it in a plasmid or a phage. Multiple labelling of such poly-DNAprobes with polymeric lanthanide chelates will therefore be anattractive procedure making it possible to overcome the disadvantagesconnected with labelling and amplifying the signal as in cases whereother non-radioactive markers are used.

The native molecule of a nucleic acid has to be modified by introducinggroups which are able to selectively react with a bifunctional couplingreagent. An example of a group which fulfills these criteria is thethiol groups. Thiol groups which can be introduced by employing severalexisting procedures (Example 6). The procedure of Example 6 is rapid,reliable, safe and should allow inexpensive small or large scalelabelling of any DNA with lanthanide chelates.

The already mentioned low reactivity of natural amino functions in DNApermits only slight cross-linking between DNA and polymeric chelates.This is necessary for persistence of the intact fragments in DNA whichare vital features of efficient hybridization. Recently there is atendency to employ short oligonucleotides instead of longer poly-DNAfragments as hybridization probes. This is especially important in thedesign of routine assays when time plays an important role. These smallfragments with the sequence long enough to be specific for the sequenceto be detected should contain at least 16 nucleotides. Sucholigonucleotides can be easily synthesized by employing commerciallyavailable reagents and apparatus, even by non-chemists. An additionalfeature of synthetic DNA fragments is their specific and regioselectivederivation in a protected state. One of these reactions is the recentlypublished procedure for selective 5'-thiolation of syntheticoligonucleotides (Example 7). This procedure, in conjunction with thealready existing procedures of specific terminal derivatization even ina fully deprotected state, together with simple preparative purificationmethods make them an alternative to the polydeoxynucleotide probes(Examples 8 and 9). Perhaps the greatest advantage using short DNAfragments becomes clear when considering the hybridization process.

Oligo DNA probes are characterized by their very favorable kinetics ofhybridization which allow the assay to proceed at low temperature and ina much shorter time period than the temperature and time periods whichare necessary for poly-DNA probes. This is of course, only true ofrelatively free oligo-DNA probes. The oligo DNA probes which are boundto globular molecules (e.g. proteins) may behave very differently and,in the most drastic case, can even totally lose their base pairingproperties. Fortunately, however, the cross-linking of 5'-functionalizedoligo-DNA probes to the linear polymers was successful without anydetectable differences in hybridization efficiency as compared to freeprobes.

Rare Earth Chelates

Different types of functionalized rare earth chelates are known in theart. Some of them exhibit fluorescence when excited at the appropriatewave length. Others do not. The fluorescent property of rare earthchelates is not critical for their use in heterogenous hybridizationassay variants of the present invention, because techniques have beendeveloped that can transform non-fluorescent rare earth chelates tofluorescent ones. Hemmila et al., Anal. Biochem. 137 (1984) 335-43). Forhomogenous hybridization assay variants, it is more critical thatchelates having fluorescent properties be selected. It is thus moreimportant to select chelates according to the stability that is requiredin the hybridization assay, than to have inherent fluorescent propertiesas the selection criteria.

In order to determine if a given chelate has the satisfactory stabilityfor use in a hybridization assay, it should be tested in the assay to berun. If the sensitivity is satisfactory the chelate is of satisfactorystability. Suitable chelates to be used in the invention havecarboxylate, phosphate or phosphonate anionic groups and/or primary,secondary or tertiary amino nitrogen atoms located in the molecule suchthat they simultaneously coordinate, via their negatively charges oxygenor nitrogen, respectively, to the rare earth metal ion so that more thanthree, preferably more than four, five- or six-membered rings areformed. This definition means that the chelates in question have morethan four, preferably more than five heteroatoms selected among theabove-mentioned nitrogens and oxygens, and that the rare earth metal ionis a joint member for all the rings. Nitrogens in aromatic rings arepresent as tertiary nitrogen atoms. Five-membered rings are preferred.This definition of a stable chelate can be found in common text-booksand includes those given in the patent literature set forth below.

Different types of chelates that can be used in our invention have beendescribed previously (EP-A-195, 413; EP-A-139,675; EP-A-68,875;EP-A-203,047; EP-A-171,978; EP-A-2,570,703; U.S. Pat Nos. 4,352,751 and3,994,466). With respect to hybridization assays that require extremelyharsh conditions we have developed a series of preferred chelates arerepresented in Example 10. They have as the common denominator apyridine ring substituted at the 2 and 6 positions with groups thattogether with the pyridine nitrogen, can chelate a metal ion. To thepyridine ring is bound only hydrogens and/or aliphatic carbon atoms.These extremely stable chelates agree with the definition for stablechelates given in text-books.

The chelate employed in the present hybridization assay has to besufficiently stable under assay process steps which requires harshconditions, such as elevated temperatures (above 60° C.) and thepresence of other chelating agents (EDTA etc.). To each linear polymermolecule may be bound more than 10, such as more than 25 chelatinggroups. The substitution degree with respect to the lanthanide chelategroups in the lanthanide chelate polymer is usually more than 20% andcan in many cases exceed 50%.

EXAMPLE 1 Preparation of Amino Functionalized Polyacrylamide (Scheme 1)

2.0 g of polyethyl acrylate (M.w. 72000--Aldrich) was treated at 50° C.with 50 ml of dry ethylenediamine in a 100 ml round bottom flask using aslow speed magnetical stirrer. After 24 h stirring the mixture wasevaporated to dryness on a rotavapor using an oil pump, and coevaporatedthree times with n-butanol. The residue after dissolving in 10 ml ofmethanol was acidified with 5 M HCl and again evaporated to dryness. Thesolid crude product was dissolved in water (20 ml) and precipitated fromacetone. Elementary analysis of the material showed a minimum of 80%conversion of starting ester functions.

EXAMPLE 2 Preparation of Carboxymethylated Polyvinylamne (CMPVA) (Scheme2)

1.0 g (12.6 mmole) of polyvinylamine hydrochloride (PVA HCl) wasdissolved in 20 ml of water and the pH of solution was brought up to10.5 by addition of 5 M NaOH. 10.5 g of bromocetic acid (75.6 mmole)dissolved in 20 ml of water and neutralized with NaOH was dropped intomagnetically stirred PVA solution, maintaining pH 10.5 by addition of 5M NaOH. After complete addition the mixture was left stirred overnight.The crude product was isolated by addition of 500 ml of ethanol. Thewhite precipitate was separated, dissolved in water and dialyzed againstdistilled water.

EXAMPLE 3 Preparation of Functionalized Polychelates by thePolvmerization of Monomeric Units (Scheme 3 and 4)

A) Synthesis of acrylamido chelate (scheme 3). 100 mg of an aminofunctionalized lanthanide chelate (EP-A-139,675) was dissolved in 5 mlof 1 M trimethylammonium bicarbonate buffer pH 9.5 and cooled to 0° C.To this stirred solution 1.5 ml of acrylol chloride was injected in fewportions. Progress of the reaction can be monitored on TLC usingacetonitrile--H₂ O (4:1) as solvent. The mixture was stirred for 1 hrand evaporated to dryness on a rotavapor, followed by severalcoevaporations with water. The final residue was dissolved in a smallamount of water and lyophilized in high vacuum.

AA) Analogously the chelates of example 10 (compounds 12) can be used.

B) Synthesis of Monoacylamido Derivative of Diaminoalkane--GeneralDescription (Scheme 4).

Monotrifluoroacetate of a (α,ω) diaminoalkane was dissolved in drypyridine: dichloromethane (1:1) and cooled to -10° C. To the stirredmixture 1.5 ml acryloyl chloride was added. The mixture was stirred for30 min. and partitioned between CHCl₃ and saturated NaHCO₃. The organicextracts were evaporated and coevaporated three times with toluene. Theoily residue was dissolved in methanol (10 ml/mmole) and an equal volumeof saturated Na₂ CO₃ was added at once. The turbid mixture becomes clearafter about 1 hr and the hydrolysis of the trifluoroacetamido group wasvirtually complete after 5 h. The reaction mixture was then evaporatedto a small volume and extracted six times with CHCl₃ /EtOH (1:1)addition of saturated (NH₄)SO₄. After evaporation the extract waspurified by short column chromatography using CHCl₃ /EtOH (6:4) as thefinal solvent. After evaporation of proper fractions the oily productwas stored at -20° C. after addition of a polymerization inhibitor.

C) Method for synthesis of functionalized polymeric chelates.

To a mixture consisting of acrylamido chelate (point A), functionalizedacrylamide (point B), in a ratio of 2:1 and dissolved in phosphatebuffer (μ=0.01) pH 8.0, so that the concentration of acrylamido monomerswas 5%, TEMED (5% aqueous solution) and ammonium persulfate (5% aqueoussolution) were added to achieve a final concentration of 0.047% (TEMED)and 0.033% (ammonium persulfate). The mixture was kept at 50° C. for 30min, and the polymer was separated from low molecular weight componentsby gel chromatography.

Using this method, polymers with chelate functions at up to 60% of itsamido groups can be easily obtained. Including other acryl monomers(e.g. acrylated amino acids or acrylated taurine) and varying the ratiosof added monomers allows for polymers of different constitution to besynthesized.

CC) By using the acrylamido chelate of step AA, other functionalizedpolyacrylamides containing the corresponding chelate can be obtained.

EXAMPLE 4 Application of Carboxymethylated Polyvinylamine (CMPVA) forthe Synthesis of Activated Polymeric Chelate (Scheme 5)

5.85 mg (0.01 mmole) of Eu³⁺ chelate in amino form (same as in Example3A) was dissolved in 50 μl of water. To this solution a solution of 2.0mg (0.02 mmole--based on carboxylate) carboxymethylated polyvinylaminein 30 μl of water was added. Solid EDAC (38 mg--0.20 mmole) was thenadded in three portions during 1 hr, and the pH of the solution wasmaintained at 5.5 by addition of diluted HCl. To this reaction mixtureof bifunctional reagent (see scheme 5), (0.32 mg--0.001 mmole), wasadded, followed by EDAC (19 mg--0.01 mmole). The mixture was incubatedfor 30 min, and the product was precipitated by addition of acetone. Thesolid, centrifuged material was dissolved in water and the polymericproduct was separated by filtration through a Trisacryl column (2×50 cm)using 50 mM Tris-HCl, 0.5 NaCl buffer pH 7.0.

The determination of the europium content in pooled high molecularfractions established a 60% incorporation of the starting monomericchelate, which corresponds approximately to 200 chelates per polymericmolecule. Finally, the material was dialyzed with water, precipitatedfrom acetone and stored as a dry powder.

EXAMPLE 5 Application of Polyvinylamine for the Synthesis of ActivatedPolymeric Chelate (Scheme 6)

Step A. 500 μg (6.3 μmole based on amino function) of polyvinylaminehydrochloride, dissolved in 20 ml of H₂ O, was labelled with 10 μmole ofan isothiocyanate functionalized lanthanide chelate (see EP-A-139,675and compound 13 of Example 10) in the presence of 10 μmoles oftriethylamine. The reaction mixture was incubated overnight at 20° C.The unreacted chelate was removed by gel filtration through a Sephadex®G-50 column (0.7×20 cm). Fractions containing lanthanide ions werepooled, desalted and concentrated. This procedure gave a polymer with ahigh degree (50-75%) of substitution.

Step B. The polymeric chelate (from Step A) was dissolved in 50 μl ofphosphate buffer pH 6.5 and a bifunctional coupling reagent (see scheme6), 270 micrograms (0.63 micromole) in 10 μl of ethanol, was added. Thereaction mixture was incubated for 6 hr with occasional shaking. Theproduct was isolated after filtration through a Sephadex® G-50 columnand precipitation from acetone.

EXAMPLE 6 Construction of Lanthanide Labelled Polydeoxyribonucleic Acid

Hybridization Assay Variant A:

10 μg of single-stranded phage M 13 mp 10 DNA containing an Adenovirus 2Xba I restriction fragment (0-3.85 arbitrary units) of about 1350 bp isreacted with 150 μl of 25% glutardialdehyde and 50 μg of aneuropium-labelled PVA (from Example 5) in phosphate-buffered saline (10mM Na--K-phosphate pH 7.4, 0.18 M sodium chloride). The reaction isallowed to proceed for 20 minutes at 37° C. After incubation thenon-reacted aldehyde groups were blocked by reacting with 500 μl of 1 Mlysine, pH 7.5 at 20° C. for one hour.

Hybridization Assay Variant B

Step A: 200 μg of single-stranded M 13 DNA containing a fragment ofAdenovirus 2 DNA was modified with sodium bisulfite-ethylenediamine atpH 6.5 for 2 h according to published methodology. The dialyzed andconcentrated sample was then dissolved in 1 ml of PBS (phosphatebuffered saline) (pH 8.5). Solid S-acetylmercaptosuccinic anhydride (1mg--5.75 μmole) was added and the reaction mixture was kept at roomtemperature (R.T.) for 60 min with occasional shaking. The mixture wasmade 0.3 M with respect to sodium acetate and 2.5 volumes of ethanol wasadded to precipitate DNA. The precipitate was dissolve din 1.0 ml of0.01 M sodium hydroxide and kept for 60 min at R.T. for hydrolysis ofS-acetyl protecting groups after which the mixture was made neutral with0.1 M HCl. DNA was precipitated and purified from low-molecular weightcompounds by gel chromatography through Sephadex® G-25 (0.7 cm×15 cm) in0.05 M phosphate buffer, pH 6.5.

Step B: 200 μg of the purified single-stranded DNA containing freesulphydryl groups and 500 μg of Eu-PVA from example 4 or example 5, stepB, were mixed and incubated at room temperature for 20 hours. TheEu-PVA-DNA conjugate was purified by gel filtration through a Sephadex®G-150 column (0.7 cm×47 cm). The column was equilibrated and eluted withPBS.

EXAMPLE 7 Construction of Lanthanide-Labelled Oligodeoxribonucleic Acid(Scheme 7)

Step A. A hexadecamer DNA probe was synthesized in large scale employingstandard solution chemistry. The 5'-protecting group was then removedand the probe was labelled with a protected thiol function (S-Tr)according to the procedure of Connolly B.A., Nucl. Acids Res. 13 (1985)4485-4502 modified by using phosphotriester chemistry, followed bystandard deprotection and purification methods. Finally the S-Trprotecting group was removed with silver nitrate in buffered by sodiumacetate system. The excess of silver was removed with hydrogen sulphideand the precipitated silver sulphide was filtered out with help of 2μfilters. The clear filtrate was concentrated and desalted on Sephadex®G-25 (10×200 mm).

Step B. 1.0 mg of 5'-thiolated oligo DNA probe was mixed with activatedPVA chelate (from Ex. 5) at pH 7.0 and in different ratios (3:1-20:1).All mixtures were incubated overnight and separated using a SephadexG-100 column (0.7 cm×50 cm). The column was equilibrated and eluted withPBS.

EXAMPLE 8 Hybridization Using DNA Probe of Long Sequence

Step A: Adenovirus 2 DNA and PBR 322 as a control DNA were denatured andspotted onto nitrocellulose filters. Amounts of DNA from 100 ng down to1 pg were applied. Filters were baked in a microwave oven for 3 minutes.

Step B: Filters obtained in Step A were prehybridized at 42° C. for 2hours in 50% formamide containing 1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH7.0, 5×Denhardt's reagent

Step C: After prehybridization the filters were transferred to ahybridization solution containing 50% formamide, 1 M NaCl 1 mM EDTA, 10mM Tris-HCl, pH 7.0, 5×Denhardt's reagent, 0.5% sodium dodecyl sulphate(SDS) and 50 μg per ml of denatured herring sperm DNA. Eu-PVA-DNAconjugate (Examples 1, 2 and 3) was added to give the final probe a DNAconcentration of 0.2 microgram/ml. The filters were hybridized at 42° C.for 4 hours. The filters were then washed at 42° C. with 0.15 M NaCl, 10mM Tris-HCl pH 7.0 containing 0.5% SDS three times for 15 minutes.

Step D: The spots in the filters were punched and the time-delayedfluorescence from europium in each spot were measured using enhancementsolution (Wallace Oy, Finland). Sensitivity of the test is 10 pg ofAdenovirus 2 DNA. Values which are twice the mean of the negativecontrols or more were considered positive.

EXAMPLE 9 Hybridization Using Oligometer DNA Probe

Step A: As in example 8.

Step B: Filters were presoaked at 30° C. for an hour in 1 M NaCl, 1 mMEDTA, 10 mM Tris-HCl pH 7.0, 5×Denhardt's reagent and 50 μg per ml ofdenatures herring sperm DNA.

Step C: After prehybridization the filters were transferred to ahybridization solution containing 1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH7.0, 0.5% SDS, 5×Denhardt's reagent and 50 μg per ml of denaturedherring sperm DNA. A Eu-PVA-hexadecamer conjugate from example 7, stepB, was added to give a hexadecamer concentration of 20 ng/ml.Hybridization was carried our at 30° C. for 3 hr. The filters werewashed in 1 M NaCl containing 10 mM Tris-HCl pH 7.0 and 0.5% SDS firstat 30° C. for 15 minutes and then at 35° C. for 10 minutes.

Step D: Spots were punched and the europium in each spot was measured asin example 8, step D. A positive signal was detected when the spots inthe filters contained 200 pg or more of Adenovirus 2 DNA.

EXAMPLE 10 Synthesis of Novel Functionalized Chelates

For a survey of the synthetic route employed and structures of thecompounds involved see scheme 8. NMR-spectra were recorded for the endproduct and intermediates synthesized and found to be in accordance withthe structures given. The compound numbers refer to those given inscheme 8.

Compound 2: Liquid ammonia (150 ml) was introduced to a 250 mlthree-necked round bottomed flask equipped with a mechanical stirrer,dropping funnel and outlet tube and immersed in a dry ice/ethanol bath.Sodium amide was generated by addition of 20 mg of iron nitrate (Fe³⁺)followed by metallic sodium (2.09 g 0.09 mole). The deep blue solutionwas stirred for one hour and the solution of collidine (1) (10.06 g,0.083 mole) in 20 ml of dry diethylether was introduced into thereaction--addition time 15 min. The formed yellow suspension was stirredfor an additional 45 min followed by the addition of benzylchloride(6.33 g, 0.05 mole) dissolved in 10 ml of dry diethylether. The reactionmixture was stirred for 45 min and the excess of sodium amide wasneutralized by addition of ammonium chloride (4.82 g, 0.09 mole)dissolved in 20 ml of H₂ O. Ammonia was evaporated and the residue waspartitioned between water and diethylether. The collected etheral phasewas dried over sodium sulphate and evaporated. The brown residual oilwas fractionated collecting a fraction distilling at 130° C./0.1 mmHg.Yield=6.17 g (55%), oil.

Compound 3: Compound (2) (20 g, 88.9 mmoles) was dissolved in THF (150ml), and nitric acid (6.7 ml, 60% aq. solution, 1 eq) was added.Diethylether was added to the clear solution until it remained dimmy,and the mixture was left in the freezer for crystallization. The whitecrystals of nitrate (quantit. yield) were added in small portions towell-chilled sulphuric acid (150 ml) never allowing the temperature toreach 10° C., whereafter the mixture was warmed at 50° C. for 10 min.The resulting brown solution was poured onto ice and neutralized withsolid sodium hydrogen carbonate. The organic material was extracted withchloroform (3×200 ml), and after drying over sodium sulphate, thechloroform extract was flash chromatographed using 4% ethanol/chloroformas a solvent. The appropriate fractions were collected and evaporatedyielding a solvent. The appropriate fractions were collected andevaporated yielding a pure yellow solid. Yield: 22.82 g (95%).

Compound 4: Compound (3) (22.82 g, 84.5 mmole) was dissolved inchloroform (100 ml), and 16 g (94 mmole) of m-chloroperbenzoic acid(mCPBA) was added in small portions at RT over a period of 30 min. Themixture was stirred for an additional 2 h and after a negative TLC testfor the starting material it was worked up by partitioning between sat.sodium hydrogen carbonate and chloroform. The combined chloroformextracts (3×200 ml) were evaporated yielding a light yellow solidmaterial that was TLC pure. Yield: 24.55 g (100%)

Compound 5: Compound (4) (24.0 g) was suspended in 100 ml of aceticanhydride. The mixture was refluxed for 20 min which resulted in ahomogenous dark solution. Acetic anhydride was evaporated on a Rotavaporand the oily residue was neutralized with saturated sodium hydrogencarbonate, followed by extraction with chloroform (3×200 ml). Thechloroform phase was evaporated and the crude material was flashchromatographed using 2% ethanol/chloroform as a solvent. The purefractions were evaporated yielding oil that was TLC and NMR pure. Yield:19.55 g (69%).

Compound 6: Compound (5) (19 g, 60.5 mmole), was oxidized as describedin Example 3. The crude, single spot on the TLC product was isolatedafter standard work up. Yield: 18.97 g (95%), oil.

Compound 7: Compound (6) (18.5 g, 56 mmole), was converted to product(7) in a synthesis analogous to the synthesis in Example 4. Theneutralized, end-extracted product was evaporated and flashchromatographed using 2% ethanol/chloroform as a solvent. The purefractions containing the product were combined and evaporated. Yield:12.04 g (61%), oil.

Compound 8: Diacetate (7) (12.0 g, 34.1 mmole), was dissolved in 50 mlof ethanol. To this solution stirred at RT, sodium hydroxide 5 M, 20 mlwas added at once. After 10 min, when the TLC test for the substrate wasnegative, the mixture was neutralized with citric acid, and partitionedbetween sat. sodium hydrogen carbonate and ethanol/chloroform 1:1. Theextraction was repeated three times using 100 ml of organic solvent foreach extraction. The combined extracts were evaporated and the residualmixture was flash chromatographed using finally 8% ethanol/chloroform asa solvent. The appropriate pure fractions were collected and evaporated.Yield: 4.75 g (52%), yellow solid.

Compound 9: To the dihydroxy compound (8) (2.7 g, 9.44 mmole), in 35 mlof dry dichloromethane, phosphorus tribromide (3.63 g, 1.26 ml, 13.41mmole) was added and the mixture was refluxed for 15 min. The reactionmixture was neutralized with saturated sodium hydrogen carbonate andextracted with chloroform (3×50 ml). The combined extracts wereconcentrated and crystallized from ethyl acetate. Yield: 3.91 g(84%)--white crystals.

Compound 10: Compound (9) (3.27 g, 7.9 mmole) and iminodiacetic aciddiethylester (5.78 g, 30.5 mmole), were coevaporated together withtoluene and redissolved in dry acetonitrile (50 ml). Solid sodiumcarbonate (10 g) was added and the mixture was refluxed for 2 h,whereafter the salts were filtered out and the filtrate was evaporated.The residue was flash chromatographed and the fractions containing theproduct evaporated to dryness. To achieve material free from anyco-chromatographed iminodiacetic acid diethylester, the oily product wastriturated with petrolether (3×20 ml) which yielded material free fromany contaminations. Yield: 5.09 g (80%), oil.

Compound 12: To the solution of compound (10) (4.8 g, 7.5 mmole) in 50ml of ethanol, 10% palladium on carbon (100 mg) was added followed bysodium borohydride (378 mg, 10 mmole). The reaction mixture was stirredat RT for 5 min and partitioned between sat. sodium hydrogen carbonateand chloroform. The chloroform extracts (3×50 ml) were concentrated andflash chromatographed to give the reduced form of compound (10)(=compound (11)) as an oil after evaporation. Yield: 3.89 g (85%).

The reduced form of compound (10) (250 mg) in 20 ml of ethanol, wastreated with 1 M sodium hydroxide (10 ml) at RT for 3 h. The product,(pure according to TLC, solvent system acetonitrile/water 4:1) wasneutralized with 1 M hydrochloric acid and concentrated. To the residuedissolved in water (25 ml), europium chloride hexahydrate (60 mg)dissolved in 5 ml of water was added and the mixture was stirred for 30min. The excess of europium salt was removed by raising the pH to 8.5with saturated sodium carbonate solution and filtration of theprecipitate. The clear solution was evaporated almost to dryness and(12) was precipitated by addition of 10 ml of acetone. The product waswashed on the filter with acetone and dried.

Compound 13: To the amino chelate (12) (100 mg) dissolved in 5 ml ofwater and vigorously stirred, thiophosgene (80 μl) dissolved in 3 ml ofchloroform was added at once and the mixture was stirred at RT for 1 h.

The water phase was separated, extracted with chloroform (3×3 ml) andconcentrated to a volume of 0.5 ml. Addition of ethanol (10 ml)precipitated (13) quantitatively as white solid. The TLC (SystemAcetonitrile/H₂ O 4:1) and fluorescence developing with acetonylacetone/EtOH (1:20) showed only a single product being negative to afluorescamine test for free amines.

Compounds 11 and 13 were employed in the preceding examples. ##STR1##

We claim:
 1. A hybridization assay method for the detection of anucleotide sequence of a nucleic acid in a sample, said methodcomprising the steps: (i) contacting under hybridization conditions thesingle stranded form of the sample nucleotide sequence with a singlestranded nucleic acid probe, wherein:a) said probe has a nucleotidesequence complementary to the sequence to be detected, b) said probe hasa water-soluble polymer bearing a plurality of chelating structurescovalently linked to said polymer, wherein said water-soluble polymer isof non-nucleic acid structure and is covalently linked to saidnucleotide sequence complementary to the sequence to be detected, and c)said probe has a plurality of rare earth metal ions that together withsaid chelating structures forms a plurality of chelate groups covalentlybound to said water-soluble polymer, and said rare earth metal isselected from the group consisting of Eu³⁺, Tb³⁺, Sm³⁺, and Dy³⁺,wherein said probe and covalently bound chelate groups are stable underhybridization assay conditions: (ii) allowing a nucleotide sequence tobe detected in said sample and the nucleotide sequence of said probe toform a double stranded nucleic acid; and (iii) detecting the formationof double stranded nucleic acid containing said probe by measuring thetime-resolved fluorescence from the rare earth metal ions incorporatedas a chelate in said double stranded nucleic acid.
 2. The method ofclaim 1, wherein the said at least one metal ion is selected from thegroup consisting of Eu³⁺ and Tb³⁺.
 3. The method of claim 1, wherein thewater-soluble polymer is selected from the group of polymers having aplurality of OH-- or amino groups.
 4. The method of claim 3, whereinsaid water-soluble polymer having a plurality of OH groups is selectedfrom the group consisting of polymers having alcohol, phenol and carboxygroups.
 5. The method of claim 3, wherein the polymer is selected fromthe group of polymers consisting of polymers having a plurality of aminogroups.
 6. The method of claim 3, wherein the water-soluble polymer isselected from the group of polymers consisting of polyvinylamines,polyethyleneimines, polylysine, polysacharides, polyacrylamides, andderivatized forms of these polymers exhibiting the said plurality ofOH--or amino groups.
 7. The method of claim 1, wherein at least twonucleotide sequences complementary to the sequence to be detected arebound to the water-soluble polymer molecule.
 8. The method of claim 1,wherein at least two water-soluble polymer molecules are bound to thenucleotide sequence complementary to the nucleotide sequence to bedetected.
 9. Nucleotide sequence to which a plurality of lanthanidechelate groups are bound covalently via a water-soluble polymer, saidlanthanide being non-radioactive and selected from a group consisting ofDy³⁺, Sm³⁺, Eu³⁺ and Tb³⁺, preferably from group Eu³⁺ and Tb³⁺.